Mimivirus Gene Promoters Exhibit an Unprecedented Conservation among all Eukaryotes

The initial analysis of the recently sequenced genome of Acanthamoeba polyphaga Mimivirus, the largest known double-stranded DNA virus, predicted a proteome of size and complexity more akin to small parasitic bacteria than to other nucleo-cytoplasmic large DNA viruses, and identified numerous functions never before described in a virus. It has been proposed that the Mimivirus lineage could have emerged before the individualization of cellular organisms from the 3 domains of life. An exhaustive in silico analysis of the non-coding moiety of all known viral genomes, now uncovers the unprecedented perfect conservation of a AAAATTGA motif in close to 50% of the Mimivirus genes. This motif preferentially occurs in genes transcribed from the predicted leading strand and is associated with functions required early in the viral infectious cycle, such as transcription and protein translation. A comparison with the known promoter of unicellular eukaryotes, in particular amoebal protists, strongly suggests that the AAAATTGA motif is the structural equivalent of the TATA box core promoter element. This element is specific to the Mimivirus lineage, and may correspond to an ancestral promoter structure predating the radiation of the eukaryotic kingdoms. This unprecedented conservation of core promoter regions is another exceptional features of Mimivirus, that again raises the question of its evolutionary origin

InParanoid 7: new algorithms and tools for eukaryotic orthology analysis

The InParanoid project gathers proteomes of completely sequenced eukaryotic species plus Escherichia coli and calculates pairwise ortholog relationships among them. The new release 7.0 of the database has grown by an order of magnitude over the previous version and now includes 100 species and their collective 1.3 million proteins organized into 42.7 million pairwise ortholog groups. The InParanoid algorithm itself has been revised and is now both more specific and sensitive. Based on results from our recent benchmarking of low-complexity filters in homology assignment, a two-pass BLAST approach was developed that makes use of high-precision compositional score matrix adjustment, but avoids the alignment truncation that sometimes follows. We have also updated the InParanoid web site (http://InParanoid.sbc.su.se). Several features have been added, the response times have been improved and the site now sports a new, clearer look. As the number of ortholog databases has grown, it has become difficult to compare among these resources due to a lack of standardized source data and incompatible representations of ortholog relationships. To facilitate data exchange and comparisons among ortholog databases, we have developed and are making available two XML schemas: SeqXML for the input sequences and OrthoXML for the output ortholog clusters

Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism

Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes

The selective elimination of messenger RNA underlies the mitosis–meiosis switch in fission yeast

The cellular programs for meiosis and mitosis must be strictly distinguished but the mechanisms controlling the entry to meiosis remain largely elusive in higher organisms. In contrast, recent analyses in yeast have shed new light on the mechanisms underlying the mitosis–meiosis switch. In this review, the current understanding of these mechanisms in the fission yeast Schizosaccharomyces pombe is discussed. Meiosis-inducing signals in this microbe emanating from environmental conditions including the nutrient status converge on the activity of an RRM-type RNA-binding protein, Mei2. This protein plays pivotal roles in both the induction and progression of meiosis and has now been found to govern the meiotic program in a quite unexpected manner. Fission yeast contains an RNA degradation system that selectively eliminates meiosis-specific mRNAs during the mitotic cell cycle. Mmi1, a novel RNA-binding protein of the YTH-family, is essential for this process. Mei2 tethers Mmi1 and thereby stabilizes the transcripts necessary for the progression of meiosis

Evolutionary Convergence on Highly-Conserved 3′ Intron Structures in Intron-Poor Eukaryotes and Insights into the Ancestral Eukaryotic Genome

The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3′ consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3′ splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures

Substrate specificity of the TRAMP nuclear surveillance complexes

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components

Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination

Combined experimental and computational approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinalis

Non-protein-coding RNAs represent a large proportion of transcribed sequences in eukaryotes. These RNAs often function in large RNA–protein complexes, which are catalysts in various RNA-processing pathways. As RNA processing has become an increasingly important area of research, numerous non-messenger RNAs have been uncovered in all the model eukaryotic organisms. However, knowledge on RNA processing in deep-branching eukaryotes is still limited. This study focuses on the identification of non-protein-coding RNAs from the diplomonad parasite Giardia intestinalis, showing that a combined experimental and computational search strategy is a fast method of screening reduced or compact genomes. The analysis of our Giardia cDNA library has uncovered 31 novel candidates, including C/D-box and H/ACA box snoRNAs, as well as an unusual transcript of RNase P, and double-stranded RNAs. Subsequent computational analysis has revealed additional putative C/D-box snoRNAs. Our results will lead towards a future understanding of RNA metabolism in the deep-branching eukaryote Giardia, as more ncRNAs are characterized

Aberrant Herpesvirus-Induced Polyadenylation Correlates With Cellular Messenger RNA Destruction

Inhibition of host cell gene expression by the human herpesvirus KSHV occurs via a novel mechanism involving polyadenylation-linked RNA turnover

Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 families

Abstract Introduction: A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers. Methods: GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene. Results: The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer. Conclusions: Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction